Majority of Escherichia coli does not cause diseases, but exists in intestinal tract of healthy human or animals without any causing particular diseases. However, some of Escherichia coli has pathogenicity, and thus causes gastrointestinal infectious diseases such as abdominal pain, vomiting, diarrhoea and the like, as well as non-gastrointestinal infectious diseases such as urethritis, cystitis, meningitides and the like. Escherichia coli that causes gastrointestinal infectious diseases orally develops infection to exhibit pathogenicity in intestinal tract. Thus, such Escherichia coli is designated as diarrhetic Escherichia coli, or as pathogenic Escherichia coli in its broader definition. Pathogenic Escherichia coli can be classified into the following 5 groups:    1) Enterotoxigenic Escherichia coli (ETEC);    2) Enteroinvasive Escherichia coli (EIEC);    3) Enteropathogenic Escherichia coli (EPEC);    4) Enterohemorrhagic Escherichia coli (EHEC); or Verotoxin-producing Escherichia coli (VTEC);    5) Enteroadherent Escherichia coli (EAEC).
O-157:H7 (hereinafter referred to as merely “O-157”) belongs to Enterohemorrhagic Escherichia coli. O-antigens have been classified into 1 to 173 types according to the differences of antigenic structures of lipopolysaccharides (LPS) that are residing on cell surfaces, while H-antigens have been classified into 1 to 57 types according to the differences of antigenic structures of flagellum. O-157 has eminently potent infectivity among causative microbes of food poisoning, thus approximately 60-80% of Enterohemorrhagic Escherichia coli is responsible for O-157. Although many of the causative microbes of food poisoning do not cause food poisoning unless from hundred of thousands to a million of cells were ingested, O-157 can result in onset of food poisoning with only several tens to several hundreds of cells. Latent period of food poisoning caused by O-157, i.e., from 4 to 10 days, is considerably long compared to those caused by the general causative microbes. Upon infection by O-157, symptoms such as diarrhoea, nausea, abdominal pain or the like, which are not distinguishable from the cases in common food poisoning, may be often developed. Further, in some cases, such symptoms as in common cold may be presented, in which disease states for example, fever, infectious manifestation in upper respiratory tract may be caused.
Verotoxin that is produced by O-157 (hereinafter referred to as “VT”; toxin which leads to lethal changes in Vero cells that are derived from renal tubular cells of African green monkey) is a similar toxin to Shigella dysenteriae toxin. VT consists of subunit A and subunit B, wherein subunit A exhibits a toxic activity, while subunit B is a part that exhibits a binding activity to mucous membranes. Genetically, VT is revealed to comprise at least 6 types, however, verotoxin 1 (hereinafter referred to as “VT1”; a toxin having almost identical amino acid sequence with that of Shigella dysenteriae toxin; MW: about 70,000) and verotoxin 2 (hereinafter referred to as “VT2”; a toxin having about 60% homology to Shigella dysenteriae toxin; MW: about 46,000) are predominantly involved in human infections. There are three types of O-157, i.e., one producing only VT1, other producing only VT2, and another producing the both. Among these types, the last type that produces both of the toxins is known to exist in a relatively large extent.
After O-157 invades into human gastrointestinal tract, it resists against gastric acid of pH 2-3, followed by early-stage colonization on epithelial cells of large intestine. Then, the bacteria externally secrete a gene product of attaching and effacing B (eae B) to colonize on cell surfaces via intimin, a gene product of eae A. This process is proposed as an attaching and effacing (A/E) lesion model [Ken-ichi Nagayama et al, Emerging infectious disease; Journal of the Japanese Society of Internal Medicine 86(11), 37, 1997]. VT binds to a Gb3 (a neutral glycolipid called globotriaosylceramide, having a structure of galactose α1-4 galactose β1-4 glucose-ceramide) receptor [Newburg, D. S. et al, J. Infect Dis., 168, 476, 1993], thereafter, it is incorporated within the cells via a coated vesicle, and then transported to endplasmic reticulum by an intracellular membrane transportation system (a trans-Golgi network) [Sandvig, K. et al., Nature, 358, 510, 1992]. Next, subunit Amoves into cytoplasm passing through a lipid bilayer to exert the toxicity. The subunit A of VT has the same RNA N-glycosidase activity as of ricin, i.e., a potent phytotoxin which is derived from seeds of Ricinus communis. It can hydrolyse an N-glycoside bond of adenosine from 5′-end at 4324th of 28S ribosome that is a member of 60S ribosome of eucaryote. Consequently, binding of aminoacyl tRNA to ribosome is inhibited, leading to cell deaths through inhibition of the protein synthesis. Moreover, it is also reported that subunit A is hydrolysed to be degraded into A1 and A2 during a transportation step predominantly to a trans-Golgi network, thus resulting in enhancement of the activity (Garred, O. et al., Exp. Cell Res., 218, 39, 1995).
Approximately 10% of enterohemorrhagic colitis may be accompanied by serious complications such as hemolytic uremic syndrome (hereinafter referred to as “HUS”), thrombotic thrombocytopenic purpura, encephalopathy and the like. HUS is an acute renal failure commonly developed during infantile period as a manifestation of thrombotic microangiopathy. Although the cause of this disease may involve enterohemorrhagic colitis, infection by pneumococci, inheritance, drug, collagen disease or the like, HUS involved in infection by O-157 has been most frequently observed. In particular, vascular endothelial cells of renal microartery, capillary of glomerulus or the like may be compromised. In addition, central neuropathy in HUS stands the first of the cause of death for HUS these days. It is reported that upon intravenous administration of VT to an animal, vascular endothelial disorder of cerebrum and cerebellum, and disorder of Purkinje cellular injury may be resulted, as well as edema and hemorrhage of nerve cells in brain stem or spinal cord (Mizuguchi, M. et al., Acta Neuropathol., 91, 254, 1996; Fujii, J. et al., Infect Immun., 62, 3447, 1994; and Fujii, J. et al., Infect Immun., 64, 5043, 1996). Accordingly, the cause may be speculated to be involved in direct or indirect influences on nerve cells, glial cells or the like through edema of vascular endothelial cells, narrowing of intravascular hollow space, platelet thrombi, fibrin thrombi, and/or further disruption of vascular brain barrier by VT.
Main factor of vascular endothelial disorder in HUS is assumed to be VT, an extracellular toxin. Among the VTs, VT2 has been known to be involved in the onset of more serious HUS (Ostroff, S. M. et al., J. Infect. Dis., 160, 994-998, 1989). However, in recent years, cytokines such as tumor necrosis factor (TNF-α), interleukin 10 (IL-1β or the like, which were produced from monocytes, macrophages, vascular endothelial cells and the like due to VT or intracellular toxin, i.e., lipopolysaccharides (LPSs), were reported to bear an important role in establishment of vascular endothelial cellular disorder (Setten, P. A. et al., Blood, 88, 174, 1996; and Tesh, V. L. et al., Infect Immun., 62, 5085, 1994). It is also reported that blood concentrations of TNF-αA and IL-1β in HUS patients during an acute phase were actually increased, and that blood TNF-α content was elevated during early stage of onset of diarrhoea in patients having VT detected in their stool (Lopez, E. L. et al, Pediatr. Infect Dis. J, 14, 594, 1995; Kar, N. C. et al., Nephron, 71, 309, 1995; and Inward, C. D. et al., Arch. Dis. Child, 77, 145, 1997).
Cytokines such as TNF-α IL-1β and the like cooperate with VT or LPS to activate polymorphonuclear leukocytes, and then accelerate their adhesion onto the vascular endothelial cells, thus resulting in disorder of vascular endothelial cells through release of active oxygen or elastase (Kar, N. C. et al., Behring Inst. Mitt., 92, 202, 1993; Morgi, M. et al., Blood, 86, 4553, 1995; Fitzpatrick, M. M. et al., Pediatr Nephrol., 6, 50, 1992; and Forsyth, K. D. et al., Lancet, 2, 411, 1989). Additionally, the expression of Gb 3 on vascular endothelial cells is increased to several hundreds folds to potentiate VT susceptibility by such actions of cytokines (Lopez, E. L. et al., Pediatr Infect Dis. J, 14, 594, 1995; Kaye, S. A. et al., Infect Immun., 61, 3886, 1993; Keusch, G. T. et al., J. Infect Dis., 173, 1164, 1996; and Forsyth, K. D. et al., Lancet, 2, 411, 1989). Furthermore, it is also reported that VT enhances the ability of leukocytes to adhere to cultured vascular endothelial cells (Morigi, M. et al., Blood, 86, 4553, 1995) and facilitates the production of endothelin (Bitzan, M. M. et al., J. Clin. Ivest., 101, 372, 1998), and thus, glomerular vascular endothelial cells that were administered with VT are compromised by peripheral leukocytes (Mahan, J. D. et al., 3rd International Symposium and Workshop on Shiga Toxin-Producing E. coli infections, p 83, Baltimore, 1997). In other reports, VT is assumed to cause necrosis (Obrig, T. G. et al., Infect Immun., 56, 2373, 1988), apoptosis (Laurence, J. et al., Semin. Hematol., 34, 98, 1997), acceleration of platelet aggregation by a von Willbrand factor (Moake, J. L. et al., Blood, 64, 592, 1984), reduction of producibility of prostaglandin 12 (Karch, H, et al., Microb. Pathog., 5, 215, 1988), deposition of fibrin onto vascular endothelial cells (Menzel, D. et al., Ann. Hematol., 68, 43, 1994), proliferation of mesangial cells and impairment of NO production (Dietrich, D. et al., J. Am. Soc. Nephrol., 1, 609, 1990) and the like, leading to onset of HUS.